Method for preparing cross-linked hyaluronic acid-based cell scaffold material

ABSTRACT

Disclosed is a method for preparing cross-linked hyaluronic acid-based cell scaffold material. The hyaluronic acid-based cell scaffold is obtained by subjecting a hyaluronic acid and a disulfide cross-linking agent to an amidation reaction, followed by dialysis-freeze drying. The cell scaffold has abundant pores, good mechanical strength which ensures that the scaffold does not rupture in transplantation, and good biocompatibility. The method is advantageous in that the raw material is easy to obtain, the reaction condition is moderate, and the process is simple. Cross-linked networks of the prepared hydrogel contain disulfide bonds, which can quickly split into single chains at the presence of small molecular glutathione. The hyaluronic acid-based cell scaffold has flexibly controllable mechanical property, disaggregation ability, and swelling property, and therefore has wide applications in facilitating cartilage injury repair, skin repair, cell culture, etc.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to Chinese Patent Application No.201810170211.8 filed on Mar. 1, 2018, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present application relates to the technical field of biomaterials,and in particular, to a method for preparing a reduction-responsivecross-linked hyaluronic acid-based cell scaffold material.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a natural mucopolysaccharide existing in human skinfor preserving moisture. It is a major component of cellular matrix andvitreous body. Hyaluronic acid is a linear polyanionicmucopolysaccharide formed by the alternate linking between (1-β-4)D-glucuronic acid (glucosamine) and (1-β-3) N-acetyl-D-glucose(glucuronic). It has an important physiological function in vivo due toits unique molecular structure and physicochemical properties.Hyaluronic acid is now widely used, mainly in the form of its sodiumsalts, in fields of cosmetics, clinical medicine, food, etc. High-purityhyaluronic acid has been widely used in ophthalmology, orthopedics, andanti-adhesion after surgery.

Hyaluronic acid can contribute significantly to the proliferation andmigration of endothelial cells in aortic and capillary, protectgranulation tissue against oxygen free-radical damage, and contribute towound healing. However, the application prospect of hyaluronic acid islimited by its weak mechanical property and rapid degradation rate invivo. For this reason, hyaluronic acid is usually modified by methodssuch as cross-linking, grafting, esterification, etc. Commonly usedcross-linking methods at present include: physical cross-linking, forexample by using sodium sulfate, sodium citrate, or sodiumtripolyphosphate; chemical cross-linking, by using a cross-linking agentsuch as 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane(DEO), divinyl sulphone (DVS), etc. These cross-linking methods controlthe cross-linking degree by regulating the concentration of thecross-linking agent and the cross-linking time, so as to prolong theexisting time of hyaluronic acid in vivo. Hyaluronic acid products usingdivinyl sulphone and 1, 4-butanediol diglycidyl ether as a cross-linkingagent have been widely used in treatment of orthopedic arthrophlogosis,operation anti-adhesion, soft tissue repair, and plastic and aestheticfilling, but their use in tissue engineering field is still beingstudied.

Due to its good biocompatibility which contributes to attachment andproliferation of seed cells, hyaluronic acid is usually used as a cellscaffold in tissue engineering. Tissue engineering requires that ascaffold material have good biocompatibility, suitable biodegradability,a three-dimensional porous structure which contributes to cellproliferation, and a certain degree of mechanical strength. When ahyaluronic acid scaffold is used to carry cells to repair a woundsurface, after the seed cells are transplanted to the wound surface tobe repaired, it would be ideal if the carrier material automaticallydegrades. However, in practice, because the hyaluronic acid aftercross-linking needs more time to degrade, if the hyaluronic acid doesnot degrade or is removed in time, it will affect therapeutic effects.Besides, the removal of the carrier may cause cell loss and secondarydamage to the wound surface. In order to solve these problems, anenvironment-responsive cell scaffold is prepared. Theenvironment-responsive cell scaffold, in response to an externalstimulus, can realize targeted delivery and release of drugs, and can becontrolled to degrade, and thus has a wide range of applications in thefuture in drug delivery and tissue repairing.

A preferred method for the degradation of hyaluronic acid isbiomacromolecular enzymolysis, which has high specificity, takes placein moderate reaction conditions, and is free of by-products. However,the limited source and high cost of hyaluronic acid enzyme restricts theapplication of hyaluronic acid enzyme. Disulfide bonds have goodstimulus-responsive property, and can effectively break into thiolgroups in a reducing condition; disulfide bonds are therefore introducedinto the structure of the cross-linked hyaluronic acid. CN101367884 andCN103613686 disclose subjecting cysteamine and hyaluronic acid to anamidation reaction to obtain thiol-hyaluronic acid hydrogel containingthiol groups, followed by dissolution of the hydrogel and an oxidationreaction between the thiol groups, to the form disulfide bonds. The mainprinciple of the foregoing is that thiol groups auto-oxidize in air toform thiol free-radicals, which collide with each other to form stabledisulfide bonds. This process is, however, very slow and it takes a longtime to form disulfide bonds. In order to accelerate the coupling ofthiol groups, CN105969825 discloses using thiol-hyaluronic acid as a rawmaterial, horseradish peroxidase as a catalyst, tyramine hydrochlorideas an enzymatic substrate, to cross-link the thiol groups by inverseemulsion method at the presence of the catalyst horseradish peroxidase,to form disulfide bonds-containing hyaluronic acid hydrogel. In thismethod, residual organic solvent and residual nonionic surfactant maylead to toxicity in the use of the material. CN104910369 disclosessubjecting an aldehyde hyaluronic acid and an amino/methylacryloylbifunctional hyaluronic acid with a side chain containing disulfidebonds to a photopolymerization reaction, to obtain cross-linkedhyaluronic acid hydrogel. The disulfide bonds can be effectivelydegraded at the presence of reducing small molecules, and thethoil-hyaluronic acid produced by the degradation can be absorbedquickly by an organism, which enables a culture system to have goodbiocompatibility.

Disulfide-containing cross-linked hyaluronic acids disclosed at presentare usually used to prepare thoil-hyaluronic acids at the presence ofdithiothreitol (DTT) for use in joint lubrication and cartilage repair.But preparation of disulfide bonds-containing cross-linked hyaluronicacid-based cell scaffolds for use in skin repair is seldom reported.

SUMMARY OF THE INVENTION

In tissue healing process, after cells migrate to a lesion area, it isrequired that a hyaluronic acid-based scaffold material graduallydegrade and maintain the microenvironment for cell growth; besides, thedegraded hyaluronic acid may facilitate the proliferation of the cells,and produce no by-product. Non-cross-linked hyaluronic acid hydrogelused as a cell scaffold has poor mechanical properties, and can causerupture of the material during transplantation, and therefore cannoteffectively carry cells for cell growth. Cross-linked hyaluronic acidhas mechanical strength that increases with the increase ofcross-linking degree, which prolongs the degradation time of the carrierscaffold, and after the migration of the cells, the residual materialmay affect skin repair.

In order to solve the problem of the incapability of controlling thedegradation of hyaluronic acid in existing technologies, the presentapplication aims to provide a method for preparing areduction-responsive cross-linked hyaluronic acid-based cell scaffoldmaterial, which, at the presence of reducing small molecules, is capableof rapidly cleaving into single-chain hyaluronic acid which can beabsorbed quickly by an organism. In order to achieve the aboveobjective, the present application adopts the following technicalsolutions:

A method for preparing cross-linked hyaluronic acid-based cell scaffoldmaterial, characterized that1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)and N-hydroxysuccinimide (NHS) are added in sequence to an aqueoussolution of hyaluronic acid having a pH value of 5.0-6.0, to form amixture; the mixture is stirred at room temperature to obtain a uniformmixture; an aqueous solution of cystamine dihydrochloride is added tothe uniform mixture to form a reaction system; the reaction system isadjusted to have a pH value of 5.0-6.0 for reaction; the reaction systemafter reaction is placed in a phosphate buffer for dialysis to form adialyzed product; and the dialyzed product is freeze-dried to obtain across-linked hyaluronic acid-based cell scaffold.

In the method for preparing cross-linked hyaluronic acid-based cellscaffold material of the present application, the carboxyl group of thehyaluronic acid is first activated, and then an amidation reaction iscarried out. The aqueous solution of hyaluronic acid is adjusted to havea pH value of 5.0-6.0, and NHS is used to activate the carboxyl group.

In the method for preparing cross-linked hyaluronic acid-based cellscaffold of the present application, the addition of cystaminedihydrochloride is performed after1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)and N-hydroxysuccinimide (NHS) has been mixed uniformly. Compared withthe direct addition of the cystamine dihydrochloride to the reactionsystem, the addition of the cystamine dihydrochloride after the uniformmixing of EDC and NHS renders the reaction system more uniform and isthus more conducive to the cross-linking reaction. The addition of theaqueous solution of cystamine dihydrochloride is preferably performeddropwise.

An advantage of the present application is that different cross-linkingcontrollable hyaluronic acids can be prepared in a one-step process tobe used as a cell scaffold. The cell scaffold contains disulfide bonds,and can cleave at the presence of a reducing agent to generatethiol-hyaluronic acid which can be quickly absorbed by an organism. Thecell scaffold also has good mucoadhesive property and in situ gellingproperty.

Further, the cell scaffold has a certain water absorption rate, and goodmechanical strength, including tensile strength, tearing strength,elongation rate at break, elastic modulus, etc. In the process ofpreparing, carrying, transferring, and transplanting, the cell scaffoldis not prone to rupture, and can be effectively used as a scaffold forcell growth.

The hyaluronic acid selected by the present application can be a metalsalt of hyaluronic acid, which is, for example, one selected from thegroup consisting of sodium hyaluronate, potassium hyaluronate, calciumhyaluronate, and magnesium hyaluronate, or a mixture of any of two ofthe foregoing, and is preferably sodium hyaluronate.

In the method of the present application, a molar ratio of —COOH in thehyaluronic acid to —NH₂ in the cross-linking agent is preferably in arange of 1:1-1:0.1, and a molar ratio of —COOH in the hyaluronic acid toEDC and NHS is preferably in a range of 1:0.1-0.6:0.1-1.

In the method of the present application, a reagent used for adjustingthe pH value is an aqueous alkali or an acid solution. The aqueousalkali includes an aqueous solution of sodium hydroxide, potassiumhydroxide, barium hydroxide, sodium carbonate, and is preferably sodiumhydroxide, more preferably 0.1 M sodium hydroxide solution. The acidsolution includes organic acid or inorganic acid, and is preferablyhydrochloric acid, more preferably 0.1 M HCl.

In the method of the present application, after the addition ofcystamine dihydrochloride, the reaction temperature is in a range of0-10° C., preferably in a range of 1-4° C.

In the method of the present application, after the addition ofcystamine dihydrochloride, the reaction time is 24-120 hours, preferably72-96 hours. Preferably, cystamine dihydrochloride is added dropwise.

In the method of the present application, before the dialysis, thehyaluronic acid hydrogel is cut into small pieces having a weight in arange of 0.2-0.5 g for dialysis.

In the method of the present application, phosphate buffer is selectedas a dialysate, and the phosphate buffer preferably contains 0.1-0.2 Mphosphate and has a pH value of 7.0-7.4.

In the method of the present application, the dialysis time is 24-72hours, preferably 48-72 hours.

In the method of the present application, the freeze-drying procedureincludes: a first pre-freezing phase, wherein the dialyzed product iskept at a temperature ranging from −60° C. to −10° C. for 2-5 hours; asecond sublimating phase, wherein the dialyzed product is kept at atemperature ranging from −40° C. to −25° C. for 4-8 hours and kept at atemperature ranging from −20° C. to 0° C. for 2-10 hours; and a thirdvacuum drying phase, wherein the dialyzed product is kept at atemperature ranging from 10° C. to 30° C. for 3-6 hours.

For the cross-linked hyaluronic acid-based cell scaffold materialprepared by the method of the present application, its three-dimensionalstructure can quickly split at the presence of a reducing substance. Thereducing small molecules selected may be dithiothreitol (DTT),glutathione (GSH) etc., and is preferably glutathione. Further, theconcentration of the reducing small molecules is in a range of 0.1-10mM.

The cross-linked hyaluronic acid-based cell scaffold material preparedby the method of the present application has a plurality ofapplications, including use as a cell scaffold for cartilage repair intissue engineering, use as a cell scaffold for skin repair in tissueengineering, and use for targeted delivery of anti-cancer drugs.

In order to prepare a cell scaffold suitable for use in skin repair, thepresent application subjects hyaluronic acid of different molecularweights and a disulfide to a cross-linking reaction, followed bydialysis, and freeze-drying, to obtain a porous cell scaffold. The cellscaffold has certain degree of mechanical strength and exhibitscontrollable splitting rate at the presence of different concentrationsof GSH, and is suitable for use in skin repair and for carrying seedcells to grow.

Compared with the existing technologies, the present application has thefollowing beneficial effects.

The method for preparing cross-linked hyaluronic acid-based cellscaffold material provided by the present application uses a smallmolecule compound containing a disulfide bond as a cross-linking agent,and subjects it and the hyaluronic acid to an amidation reaction, toobtain disulfide bond-containing cross-linked hyaluronic acid hydrogelin a one-step process. The method is advantageous in that the rawmaterial for the method is easy to obtain, the reaction conditionthereof is moderate, and the process thereof is simple. By way of theamidation reaction, the hyaluronic acid is provided with athree-dimensional network structure in which the three-dimensionalnetworks are connected to each other through chemical bonds; thehyaluronic acid therefore has good mechanical property and a uniformstructure. A porous cell scaffold is obtained by freeze-drying thehydrogel.

The cross-linked hyaluronic acid-based cell scaffold provided by thepresent application has cross-linked networks containingreduction-responsive disulfide bonds, which degrades/disaggregates inresponse to an external stimulus. The cross-linked hyaluronic acid-basedcell scaffold has a reducing disaggregation mechanism, excellentbiocompatibility, good mechanical property, structural stability,flexible and controllable disaggregation ability. The mechanicalproperty and the three-dimensional system of the cross-linked hyaluronicacid hydrogel prepared by the present application can therefore beeffectively regulated and controlled. The cross-linked hyaluronic acidhydrogel can be used as a cell scaffold for in-vitro cell culture intissue engineering, can be used in cell transplantation as well astissue and cartilage repair, and has wide applications in the future inbiomedical field.

The cross-linked hyaluronic acid-based cell scaffold prepared by themethod of the present application can be used as a cell scaffold forcartilage repair in tissue engineering, and can be used a carrierscaffold for in-vitro culture of human or animal cartilage cells, forexample for use in repair of cleft lip and palate.

The cross-linked hyaluronic acid-based cell scaffold prepared by themethod of the present application can be used as a scaffold for in-vitrocell culture for skin repair in tissue engineering, can be used for thepreparation of tissue engineered epidermis of epidermal cells,melanocytes, and fibrocytes, and for the repair of leucoderma and burnwounds.

The cross-linked hyaluronic acid-based cell scaffold prepared by themethod of the present application can be used for the carrying,delivery, and intelligent release of anti-cancer drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a route diagram of preparing cross-linked hyaluronic acidaccording to the present application; and

FIG. 2 shows reduction-responsiveness of cross-linked hyaluronicacid-based cell scaffold material prepared according to Example 2 of thepresent application.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present application could be further understood from the followingspecific embodiments, which, however, are not intended to limit thepresent application.

I. Preparation of Cross-Linked Hyaluronic Acid-Based Cell ScaffoldMaterial

FIG. 1 shows a route diagram of preparing cross-linked hyaluronic acidaccording to the present application.

Example 1

Hyaluronic acid (1 g, having 2.48 mmol of —COOH) (injectable grade,molecular weight thereof being 0.9-1.1 million) was dissolved in 100 mLof deionized water. The resultant solution was adjusted to have a pHvalue of 5.0, followed by sequential addition of 0.475 g of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and0.285 g of N-hydroxysuccinimide (NHS). The resultant mixture was stirredat room temperature for 3 hours. Cystamine dihydrochloride (0.153 g,0.99 mmol) was dissolved in 5 mL of water to form a solution, which wasadded dropwise to the reaction system. After the dropwise addition ofthe solution, the resultant mixture was readjusted to have a pH value of5.0-5.5, stirred at 4° C. to react for 96 hours. After that, theresultant mixture was placed in a phosphate buffer having a pH value of7.4 for dialysis for 72 hours, pre-freezed at −50° C. for 3 hours,maintained at −30° C. for 6 hours, maintained at −15° C. for 8 hours,and then dried at 25° C. in vacuum for 4 hours, to produce across-linked hyaluronic acid-based cell scaffold (First Group: 1-1).

Example 2

Hyaluronic acid (1 g, having 2.48 mmol of —COOH) (injectable grade,molecular weight thereof being 0.9-1.1 million) was dissolved in 80 mLof deionized water. The resultant solution was adjusted to have a pHvalue of 5.0, followed by sequential addition of 0.380 g of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and0.228 g of N-hydroxysuccinimide (NHS). The resultant mixture was stirredat room temperature for 2.5 hours. Cystamine dihydrochloride (0.115 g,0.74 mmol) was dissolved in 5 mL of water to form a solution, which wasadded dropwise to the reaction system. After the dropwise addition ofthe solution, the resultant mixture was readjusted to have a pH value of5.0-5.5, stirred at 4° C. to react for 90 hours. After that, theresultant mixture was placed in a phosphate buffer having a pH value of7.4 for dialysis for 65 hours, pre-freezed at −50° C. for 2 hours,maintained at −30° C. for 5 hours, maintained at −15° C. for 8 hours,and then dried at 25° C. in vacuum for 4 hours, to produce across-linked hyaluronic acid-based cell scaffold (Second Group: 1-2).

Example 3

Hyaluronic acid (1 g, having 2.48 mmol of —COOH) (injectable grade,molecular weight thereof being 1.7-1.9 million) was dissolved in 60 mLof deionized water. The resultant solution was adjusted to have a pHvalue of 5.0, followed by sequential addition of 0.285 g of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and0.171 g of N-hydroxysuccinimide (NHS). The resultant mixture was stirredat room temperature for 3 hours. Cystamine dihydrochloride (0.076 g,0.49 mmol) was dissolved in 5 mL of water to form a solution, which wasthen added dropwise to the reaction system. After the dropwise additionof the solution, the resultant mixture was readjusted to have a pH valueof 5.0-5.5, stirred at 4° C. to react for 85 hours. After that, theresultant mixture was placed in a phosphate buffer having a pH value of7.4 for dialysis for 60 hours, pre-freezed at −50° C. for 3 hours,maintained at −30° C. for 6 hours, maintained at −15° C. for 6 hours,and then dried at 25° C. in vacuum for 4 hours, to produce across-linked hyaluronic acid-based cell scaffold (Third Group: 1-3).

Example 4

Hyaluronic acid (1 g, having 2.48 mmol of —COOH) (injectable grade,molecular weight thereof being 1 million) was dissolved in 50 mL ofdeionized water. The resultant solution was adjusted to have a pH valueof 5.0, followed by sequential addition of 0.190 g of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and0.114 g of N-hydroxysuccinimide (NHS). The resultant mixture was stirredat room temperature for 1.5 hours. Cystamine dihydrochloride (0.038 g,0.25 mmol) was dissolved in 5 mL of water to form a solution, which wasthen added dropwise to the reaction system. After the dropwise additionof the solution, the resultant mixture was readjusted to have a pH valueof 5.0-5.5, stirred at 4° C. to react for 80 hours. After that, theresultant mixture was placed in a phosphate buffer having a pH value of7.4 for dialysis for 54 hours, pre-freezed at −50° C. for 2 hours,maintained at −30° C. for 4 hours, maintained at −15° C. for 6 hours,and then dried at 25° C. in vacuum for 4 hours, to produce across-linked hyaluronic acid-based cell scaffold (Fourth Group: 1-4).

II. Measurement of Thickness and Pore Diameter of the Cross-LinkedHyaluronic Acid-Based Cell Scaffold

Method: The scaffold material was cut into small pieces, and thicknessof the material was measured using a vernier caliper. Results showedthat with the increase of the feeding amount of the cross-linking agent,the thickness of the material gradually increased. When the content ofthe cross-linking agent was relatively high, the obtained cell scaffoldhad a uniform thickness and uniform pore diameters. But when the contentof the cross-linking agent was too high, the obtained cell scaffold hadvery small pore diameters, which was not conducive to the exchange ofnutrient substances during the culturing of cells. When the content ofthe cross-linking agent was relatively low, the cell scaffold obtainedafter freezing and drying had a non-uniform thickness, and in particularhad relatively large pore diameters (some even reaching over 400 μm) atrelatively thin locations of the scaffold, as a consequence of which thematerial had a relatively weak strength and the scaffold was thus proneto rupture. Therefore, it was concluded from the thickness and porediameter of the material that, when the proportion of the cross-linkingagent was suitable, the prepared material had a uniform pore diameterwith a suitable size, in which case the material, by taking advantage ofcell proliferation and growth, was capable of providing amicroenvironment for cell growth.

TABLE 1 Thickness and Pore diameter of the cross-linked hyaluronicacid-based cell scaffold produced in Examples 1 to 4 Molar RatioThickness Pore diameter Sample No. (—COOH:—NH₂) (mm) (μm) 1-1 1:0.8 1.14± 0.15 100 ± 16 1-2 1:0.6 1.36 ± 0.34 121 ± 38 1-3 1:0.4 1.92 ± 0.81 136± 21 1-4 1:0.2 2.03 ± 0.96 153 ± 42

III Measurement of Mechanical Strength of the Cross-Linked HyaluronicAcid-Based Cell Scaffold

Method: The cross-linked hyaluronic acid-based cell scaffold produced inExamples 1 to 4 was cut into 3×3 cm small pieces, and tensile propertyof the produced material was measured using a tensile machine. Resultsshowed that, when the proportion of the disulfide cross-linking agentwas relatively large, the prepared scaffold material had a relativelyhigh mechanical strength. The scaffold material ruptures when thetensile force was 8-9 N, which ensured that when the cross-linkedhyaluronic acid-based cell scaffold prepared by the method according tothe present application was transferred and cut in the process of celltransplantation, the material did not break or rupture; the cross-linkedhyaluronic acid-based cell scaffold could therefore be used as ascaffold for cell growth and provided a microenvironment for cellgrowth. But when the cross-linking degree was low, the scaffold materialhad a non-uniform thickness, in which case, the material ruptures atrelatively thick locations when the tensile force was 4-5 N, andruptures at relatively thin locations when the tensile force was 3-4 N.The larger the molecular weight of the hyaluronic acid was, the largerthe tensile strength thereof was, as shown by sample 1-2 and sample 1-3.

TABLE 2 Tensile strength of the cross-linked hyaluronic acid-based cellscaffold prepared in Examples 1-4 Sample No. Molar Ratio (—COOH:—NH₂)Tensile Strength (N) 1-1 1:0.8 8.5 ± 0.9 1-2 1:0.6 7.3 ± 1.2 1-3 1:0.46.8 ± 1.5 1-4 1:0.2 4.8 ± 1.8

IV Expansion Rate and Water Absorption Rate of the Cross-LinkedHyaluronic Acid-Based Cell Scaffold

Method of measuring expansion rate: The cross-linked hyaluronicacid-based cell scaffold produced in Examples 1 to 4 was cut into 3×3 cmsmall pieces. The small piece was put into 30 mL of 0.9% sodium chloridesolution, and placed in a water bath at 37° C. for 2.5 hours. The lengthand width of the piece was then measured. The expansion rate was thepercentage ratio of the length multiply by the width after the swellingto the length multiply by the width before the swelling.

Method of measuring water absorption rate: The cross-linked hyaluronicacid-based cell scaffold produced in Examples 1 to 4 was cut into 2×2 cmsmall pieces. The small piece was weighed and the weight thereof wasrecorded as W₁. The small piece was put into 20 mL of 0.9% sodiumchloride solution at 37° C. for 10 minutes, taken out using a pair oftweezers, followed by removing unnecessary moisture from its surface,and then weighed and the weight thereof was recorded as W₂. The waterabsorption rate was the ratio of the weight of water absorbed by thescaffold material during a certain period of time to the weight of thescaffold material per se.

TABLE 3 Expansion rate and water absorption rate of the cross-linkedhyaluronic acid-based cell scaffold produced in Examples 1 to 4 MolarRatio Expansion Rate Water Sample No. (—COOH:—NH₂) (%) Absorption Rate1-1 1:0.8  94.9 ± 4.3 43.2 ± 1.5 1-2 1:0.6  99.8 ± 2.9 52.3 ± 2.1 1-31:0.4 103.6 ± 1.9 56.7 ± 3.5 1-4 1:0.2 113.5 ± 6.3 78.4 ± 2.6

V Reducing Property of Cross-Linked Hyaluronic Acid Hydrogel

Method: The cross-linked hyaluronic acid-based scaffold material (1-2)was cut into small pieces, from which three parts were weighed. Eachpart weighed 10 mg. PBS solution (pH=7.4) having 0.1 mM of GSH, PBSsolution (pH=7.4) having 2 Mm of GSH, and PBS solution (pH=7.4) having10 mM of GSH were added into the three parts, respectively. Theresultant solutions were placed in a thermostatic water bath at 37° C.under magnetic stirring. After the reaction was carried out for acertain period of time, the content of free hyaluronic acid aftercleavage was determined, and the content of uronic acid was determinedby measuring and diluting 1 mL of supernatant.

GSH (glutathione) is considered to be the most importantoxidation-reduction couple in animal cells. It determines theantioxidant ability of cells. The introduction of GSH does not lead tocytotoxicity, and therefore the cleavage of the carrier scaffold doesnot adversely influence the growth of cells. The free hyaluronic acidproduced by the cleavage of the cross-linked hyaluronic acid-basedscaffold was measured under the condition that concentrations of GSHwere 0.1 mM, 2 mM, and 10 mM, respectively, and a phosphate bufferedsolution having a pH value of 7.4 was used as a control. Results areshown in FIG. 2, and indicate that under a reducing condition, thecross-linked hyaluronic acid-based scaffold can gradually decompose withthe passage of time, until it becomes a solution. In addition, thelarger the concentration of the reducing agent, the faster thethree-dimensional structure of the produced scaffold materialdecomposes. A new approach is thus provided for theremoval/decomposition of scaffold material after cells are transplantedto a wound surface in in-vitro cell culture and cell transplantation oftissue engineering study.

VI Cytotoxicity of Cross-Linked Hyaluronic Acid Hydrogel-Based CellScaffold Material

MTT Assay

In accordance with Biological Evaluation of Medical Devices—Part 5:Tests for In Vitro Cytotoxicity, the produced cross-linked hyaluronicacid hydrogel-based cell scaffold was cut into small pieces having adimension of 0.1-0.3×0.1-0.3. Then, 1 g of the small pieces was measuredand added to 1 mL of a cell culture liquid, and placed at a temperatureof 37±2° C. for 30 hours. The leaching liquid was diluted with a culturemedium, to obtain a series of diluted leaching liquids as testsolutions. Results are shown in Table 5.

TABLE 5 Cytotoxicity of cross-linked hyaluronic acid-based cell scaffoldprepared in Examples 1 to 4 Samples Cytotoxic Reactions 1-1 Grade 0 2-1Grade 0 3-1 Grade <1 4-1 Grade <1

The above results show that the cross-linked hyaluronic acid-based cellscaffold material prepared according to the present application hasexcellent properties, especially in material pore diameter, expansionrate, and water absorption rate. Furthermore, the prepared cross-linkedhyaluronic acid-based cell scaffold has good biocompatibility and goodreduction-responsiveness. Therefore, the cross-linked hyaluronicacid-based cell scaffold of the present application can be used in theculture of in-vitro cells in cartilage/skin repair, and can also be usedto carry an anti-cancer drug to a specific site for intelligent releaseof the anti-cancer drug.

The above embodiments are merely for illustrating the principles of thepresent application, and are not intended for limiting the presentapplication. Various variations and modifications can be made to thepresent application within the spirit and scope defined by the claims ofthe present application, and all such variations and modifications shallfall within the protection scope of the present application.

The invention claimed is:
 1. A method for preparing a cross-linkedhyaluronic acid-based cell scaffold material, comprising the steps of:adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC.HCl) and N-hydroxysuccinimide (NHS) in sequence to an aqueoussolution of hyaluronic acid having a pH value of 5.0-6.0, to form amixture; stirring the mixture at room temperature to obtain a uniformmixture; adding an aqueous solution of cystamine dihydrochloride to theuniform mixture to form a reaction system; adjusting the reaction systemto have a pH value of 5.0-6.0 for reaction; placing the reaction systemafter reaction in a phosphate buffer for dialysis to form a dialyzedproduct; and freeze-drying the dialyzed product to obtain thecross-linked hyaluronic acid-based cell scaffold, wherein the step offreeze-drying comprises: at a first pre-freezing phase, keeping thedialyzed product at a temperature ranging from −60° C. for 2-5 hours; ata second sublimating phase, keeping the dialyzed product at atemperature ranging from −40° C. to −25° C. for 4-8 hours and keeping ata temperature ranging from −20° C. to 0° C. for 2-10 hours; and at athird vacuum drying phase, keeping the dialyzed product at a temperatureranging from 10° C. to 30° C. for 3-6 hours.
 2. The method according toclaim 1, wherein a molar ratio of —COOH in the hyaluronic acid to —NH₂in the cross-linking agent is in a range of 1:1-1:0.1, and a molar ratioof —COOH in the hyaluronic acid to EDC and NHS is in a range of1:0.1-0.6:0.1-1.
 3. The method according to claim 1, wherein thereaction is carried out at a temperature in a range of 0-10° C.
 4. Themethod according to claim 1, wherein the reaction is carried out for24-120 hours.
 5. The method according to claim 1, wherein an aqueousalkali or an acid solution is used for adjusting the pH value.
 6. Themethod according to claim 1, wherein hyaluronic acid based cell scaffoldis cut into small pieces having a weight in a range of 0.2-0.5 g forconducting the dialysis.
 7. The method according to claim 1, wherein thephosphate buffer contains 0.1-0.2 M phosphate and has a pH value of7.0-7.4.
 8. The method according to claim 1, wherein the dialysis isconducted for 24-72 hours.
 9. The method according to claim 3, whereinthe reaction is carried out at a temperature in a range of 1-4° C. 10.The method according to claim 4, wherein the reaction is carried out for72-96 hours.
 11. The method according to claim 5, wherein the aqueousalkali is 0.1 M sodium hydroxide solution, and the acid solution is 0.1M HCl.
 12. The method according to claim 8, wherein the dialysis isconducted for 48-72 hours.